Indicator compounds, method of their preparation and use of those compounds in an iron assay system

ABSTRACT

The present invention is related to compounds of the general formula (I) ##STR1## wherein R 1  denotes hydrogen, halogen or C 1  -C 4  alkyl and 
     R 2  denotes C 1  -C 4  alkyl, aryl or heteroaryl, or a substituted C 1  -C 4  alkyl, aryl or heteroaryl and their preparation. The compounds can be used in an iron assay system.

SUMMARY OF THE INVENTION

The present invention is related to compounds of the general formula (I)##STR2## wherein R¹ denotes hydrogen, halogen or C₁ -C₄ alkyl and

R² denotes C₁ -C₄ alkyl, aryl or heteroaryl, or a substituted C₁ -C₄alkyl, aryl or heteroaryl, and their preparation. The compounds can beused in an iron assay system.

Alterations of iron metabolism reveal themselves both as deficiencydiseases and overload diseases of the element. Iron deficiency diseasesare widely diffused and also affect populations of highly developedcountries besides obviously underfed people. Iron deficiency anemiapreferentially affects women for increased iron losses withmenstruation, pregnant women for increased iron needs, patients withulcer or other causes of acute or chronic bleeding. Also neonates areparticularly exposed to the disease.

The diagnosis of iron deficiencies, their differentiation from low bloodiron during phlogistic diseases, and the monitoring of therapy, requireaccurate and reproducible laboratory tests.

The laboratory diagnosis in this field takes advantage essentially ofthe measurements of:

blood iron

blood transferrin

blood ferritin

globular volume and hemoglobin

free erythrocyte protoporphyrin.

While all the other tests are largely established in the clinicallaboratory, the free erythrocyte protoporphyrin assay is now emergingfrom the "experimental incubation" phase for the clinical routineapplication.

The term blood iron refers to the level of iron actually transported inthe plasma and consequently bound to transferrin. Although it is not asure index of the iron content of the body, the blood iron assay isvaluable to estimate the status of the amount of stored iron. Majorcauses that can lead to an iron deficiency or surplus are shown in TableI.

                  TABLE I                                                         ______________________________________                                        Causes of Variation of Blood Iron                                             ______________________________________                                        CAUSES OF DECREASE                                                            Insufficient intake of dietary iron                                           (babies, vegetarians)                                                         Defective absorption                                                          (total and subtotal gastrectomy, achlorhydria,                                chronic diarrhoea and steatorrhoea)                                           Prolonged blood losses                                                        (chronic hemorrhage due to gastric, duodenal                                  ulcer, etc.)                                                                  Increased needs                                                               (pregnancy, lactation)                                                        Iron storage in the cells of the RE system                                    (chronic or other infections)                                                 CAUSES OF INCREASE                                                            Increased degradation of erythrocytes                                         (Hemolytic anemia, autoanticorpal anemia)                                     Disorders of hemoglobin synthesis                                             (pernicious anemia, sideroachrestic anaemia)                                  Acute liver diseases                                                          (viral hepatitis, toxic hepatitis)                                            Hemosiderosis                                                                 Hemochromatosis                                                               ______________________________________                                    

Iron deficiency generally evolves through various phases slowly beforeresulting in frank anemia. It should be noted that a low blood ironlevel does not necessarily reflect the existence of a status of irondeficiency. Blood iron in addition deviates markedly from normality onlywhen the variations of the status of saturation of the amount of storediron have become significant.

The blood iron is A quite variable parameter. It presents marked andwell known fluctuations both within-a-day and between-days.

Various authors have documented the existence of a circadian rhythm ofblood iron, with a peak in the morning between 8 and 10 and lower valuesin the late afternoon. Particularly interesting is that, in subjectworking at night, the more elevated values are shifted in the afternoon,in phase with the cycle-sleep-activity; consequently the rhythm appearsto be reversed.

The reference intervals reported in the literature are also different;the normal values are, among other things, influenced by physiologicalfactors such as age (higher values in the neonate and lower values inthe elderly) and sex (slightly high values in men).

The biological variability of blood iron and the possibility ofincreases for cellular necrosis processes (for example acute liverdiseases) and decreases for phlogistic conditions (because of the bondwith transferrin) limit the diagnostic value of the measurement.

For more detailed information reference is made, for example, to "IronClinical Significance and Methods of Assay" a publication of July 1986edited by Ames Division, Miles Italiana S.p.A.

The most serious problem of iron testing is the low concentration of theanalyte especially in case of a decrease. Further the tight bond betweenthe iron and the transferrin requires drastic reaction conditions (inthe assay system) to release the iron from this transport protein.Interference caused, for example, by copper is a serious problem, too.

The present invention now provides new compounds which are very usefulas indicator compounds in an iron assay system. The complex of iron withthe present compounds not only show a high sensitivity but also a veryhigh stability at lower pH-ranges. The stability of the iron-indicatorcomplex at lower pH is very desirable, because this allows less drasticconditions for the promotion of the release of iron from the transportof the protein transferrin.

The present invention concerns compounds of the general formula (I)##STR3## wherein R¹ denotes hydrogen, halogen or alkyl and

R² denotes C₁ -C₄ alkyl, aryl or heteroaryl or a substituted C₁ -C₄alkyl, aryl or heteroaryl.

Preferred are compounds of formula (I) wherein

R¹ denotes hydrogen and

R² denotes C₁ -C₄ alkyl, aryl or thienyl, which radicals can besubstituted by C₁ -C₄ alkyl, hydrogen, halogen or SO₃ H.

More preferred are compounds of formula (I) wherein

R¹ denotes hydrogen and

R² denotes methyl, ethyl, propyl, phenyl, tolyl or thienyl.

In particular the present invention concerns compounds of formula (I)wherein

R₁ denotes hydrogen and

R₂ denotes ##STR4## wherein R¹¹ denotes hydrogen, methyl or chlorine.

The most preferred compound is a compound having the formula ##STR5##

The present invention is further related to an assay system fordetection or quantitative determination of iron in a sample comprisingone of the compounds.

In a preferred embodiment the iron assay system further comprises areducing compound of the formula ##STR6##

Consequently the present invention is also related to the use of thiscompound in the determination of iron. The mentioned quinoline compoundhas been determined as very useful for the reduction of Fe³⁺ to Fe²⁺,which is necessary in the iron test system. Ascorbic acid or otherreducing agents known in the art can also be used in connection with thenew indicators of the present invention, but the above quinolinecompound is preferred for the purpose of reduction. Consequently, thepresent invention is also related to the use of3-hydroxy-1,2,3,4-tetra-hydro-benzo(h)-quinoline as reducing agent in anassay system for the determination of iron.

The iron assay-system can furthermore contain substances which do notreact, such as, for example, buffers, wetting agents, stabilizers andthe like.

Reagent combinations can be prepared from the above compounds. Thereagent combination can be in the form of a solution or as a powder orare in the form of tablets or a lyophylisate. The reagent combination(if it is not already in the form of a solution) is taken up in water oranother suitable solvent and a reagent solution is prepared. If thereagent combination consists of individual components, these are to bemixed with one another. After the sample (for example blood, serum,plasma or urine) has been mixed with an aliquot portion of the reagentmixture, the color formed is measured on a photometer and theconcentration of iron is calculated via the molar extinction coefficientand the volumes of reagent and sample added, or via an iron standardaqueous solution.

The iron assay system can furthermore be impregnated, together with abuffer system, with appropriate wetting agents and activators as well asother auxiliaries, onto absorbent reagent carriers, such as papers,fleeces and the like. For this, one or more impregnating solutions canbe prepared in the form of aqueous, organic or mixed solutions,depending on how the reagents or auxiliaries dissolve. Absorbent orswellable carriers, preferably filter paper or absorbent fleece of glassor plastic, are impregnated or sprayed with these solutions. Thecarriers are then dried. The reagent carriers thus prepared can be usedeither as rapid diagnostics for direct determination of the contents ofthe analyte in the liquid (for example in body fluids, such as blood,urine or saliva, in foodstuffs, for example fruit juices, milk and thelike). The liquid is thereby applied directly to the reagent carrier orthis is immersed briefly in the liquid. Semiquantitative determinationis possible by allocating a comparison color to the thus formed.Quantitative evaluation can be carried out by reflectance photometry.

It is also possible to introduce the test agent according to theinvention into carrier matrices prepared from casting solutions.Examples which my be mentioned here are cellulose, cellulosederivatives, gelatin, gelatin derivatives or plastics, such aspolyurethanes and polyacrylamide. It is advantageous here that the testagent and if appropriate the other necessary reagents are added directlyto the casting solution, which means that it is possible to produce thetest device, consisting of the carrier and reagents, in one operation.

By eluting the above-mentioned reagents with water or buffer or serumfrom the absorbent carrier, a reagent solution can be prepared, withwhich the analyte or enzymes can be determined in the cell of aphotometer as described above.

Wetting agents are, in particular, anionic and cationic, nonionic oranphotheric wetting agents.

Other auxiliaries which my be appropriate are the customary thickeners,solubilizing agents, emulsifiers, optical brighteners, contract mediaand the like, such as are known in corresponding tests with otherchromogens.

The preparation of the compounds according to the invention can beillustrated by way of an example: ##STR7##

The required starting materials are known from the literature [of forexample: Acta. Chem. Scand., 23, 1087 et seq (1969); J. Amer. Chem.Soc., 75, 1115 (1953); Organikum, Organisch chemisches Grundpraktikum,page 325 et seq. (1970)].

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the average absorbance of aqueous iron solutions v.s.solution pH.

FIG. 2 shows the minimum required concentration of the presentformulation.

FIG. 3 shows a useful range of the present formulation.

FIG. 4 shows the linear relationship of the present formulation using aNBS standard up to a concentration of 1000 μg/dl of iron.

FIG. 5 shows a linear correlation between the Sera-Pak Iron test kit andthe present formulation.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Preparative Examples Example 1

6.44 g (grams) of 3-(2-pyridyl)-5,6-bis(2-thienyl)-1,2,4-triazine areintroduced into 25 ml (milliliter) of 25% strength oleum at 0° C. Thereaction mixture is allowed to reach room temperature and is stirredfurther for 24 hours. The sulphonation mixture is then discharged on to˜100 g of ice, buffered with a small quantity of NaOH and theprecipitate is filtered off with suction. 8.4 g of a yellow powder areisolated which forms a blue color with iron (II) ions in water (λ_(max)=593 nm; c=34000). It is clear from the NMR spectrum that the powder isone of the following possible isomers: ##STR8##

Example 2

The 3-(2-pyidyl)-5,6-bis (2-thienyl)-1,2,4-triazine required in Example1 is prepared in the following manner:

13.6 g of picolineamidrazone and 22.2 g of thenil are stirred in 125 mlof ethanol at room temperature. After 24 hours the reaction mixture isconcentrated in a rotary evaporator and the residue is recrystallizedfrom absolute ethanol. C - 13 NMR data

    ______________________________________                                         ##STR9##                                                                 

    ______________________________________                                               C-1          150.40 D                                                         C-2          125.33 D                                                         C-3          139.01 D                                                         C-4          124.04 D                                                         C-5          160.04 D                                                         C-6          152.63 S                                                         C-7          149.54.sup.a S                                                   C-8          ****.sup.a.S                                                     C-9          136.89 S                                                         C-10         131.73 D                                                         C-11         129.60 D                                                         C-12         128.83 D                                                         C-13         137.11 D                                                         C-14         128.27 D                                                         C-15         129.81 D                                                         C-16         132.32 D                                                  ______________________________________                                    

Example 3

284 g of phosphorous pentaoxide are stirred in a mixture of 2500 ml oftoluene and 250 ml of thiophene. Then 300 g tolylacetic acid are addedin portions at 80° C. The mixture is subsequently stirred for 5 hoursand is then discharged onto ice. The organic phase is separated off,dried and concentrated in a rotary evaporator. The residue isrecrystallized from aqueous ethanol. 326 g of the following compound##STR10## are obtained.

Example 4

27.75 g of selenium dioxide are suspended in a mixture of 250 ml ofdioxane and 20 ml of water. 54.0 g of the thienyl ketone of Example 3are added to this suspension and the mixture is then heated for 6 hoursunder reflux, filtered off by suction to remove the residue and thereaction mixture is concentrated in a rotary evaporator. 49.2 g of abrown oil of the following formula ##STR11## are obtained and processedfurther without purification.

Example 5

23.0 g of the diketone prepared in Example 4 are heated under refluxwith 13.6 of picolineamidrazone in 100 ml of ethanol. The triazineformed already crystallizes out under boiling heat. After 1 hour themixture is cooled and filtered off with suction. 33.9 g of a yellowpowder with a melting point of 191° C. are isolated. The spectra do notallow the product to be assigned definitely to one or other of thefollowing two isomers: ##STR12##

Example 6

If the triazine of Example 5 is reacted with 25% strength oleumaccording to the process described in Example 1, the monosulphonatedcompound is obtained which can be assigned to one of the followingstructures: ##STR13##

Calculated: C, 52.77; H, 3.03; N, 12.96; O, 11.1; S, 14.83; N, 5.31.

Found: C, 52.3; H, 3.1; N, 13.0; S, 15.0.

Example 7

If 6.44 g of 3-(2-pyridyl)-5,6-bis(2-thienyl)-1, 2,4-triazine is stirredin 50 ml of sulphonated monohydrate for 7 hours at 50° C. and themixture is then worked up as described in Example 1, 3.2 g of thefollowing compound, which has a melting point of higher than 250° C.,are obtained: ##STR14##

Example 8

If pyridinylthiophene is reacted with picolineamidrazone as described inExample 5 one of the two following possible triazines are obtained in ayield of 80% [JR:1385 cm⁻¹ (-CH₃)]: ##STR15##

Example 9

The triazine derivative of Example 8 is sulphonated in monohydrate at atemperature of 50° C. After working up a yellow powder which correspondsto one of the following formula, is obtained in a 65% yield. ##STR16##

Analysis:

Calculated: C, 43.82; H, 2.55; N, 15.72; O, 13.47; S, 17.99; Na 6.45.

Found: H, 44.00; H, 2.45; S, 18.2.

Test Examples

In the following iron tests the compound of Example 1 has been used asindicator.

Principle of the Test

Iron in human serum is released from its carrier protein, transferrin,in an acid medium and simultaneously reduced to the ferrous form by areducing agent. Ferrous ions chelate with the indicator forming a stableblue complex whose absorbance, spectrophotometrically read at 593 nm(nanometers), is proportional to the iron content. Deproteinization isnot required. A sample blank is required to correct for the serum matrixeffect.

MATERIALS AND METHOD

Experiments (formula optimization, linearity, comparison studies, etc.)were carried out according to the following directions:

Sample: human plasma heparinized or human sera (native or spike withferric ions) obtained from hospital routine were used. Aqueous solutionsof iron were prepared dissolving from iron metal (NBS material code 937)with nitric acid and diluting to the appropriate concentration withdistilled water.

Instrumentation: a double-beam spectrophotometer (model Lambda 5, PerkinElmer Corp.) was used.

Materials: The iron indicator was the compound of Example 1, all othercompounds were reagent-grade materials. The working solution containsthe buffer, the reducing agent, thiourea (to suppress a possible copperinterference) and the indicator compound. A working solution without theindicator compound was also prepared for the sample blank tests. Forcomparison studies the SERA-PAK Iron kit, Ferene-S method of AmesDivision, Miles Italiana S.p.A., was used.

Test procedure

    ______________________________________                                        Wavelength    593 nm (570-610)                                                Cuvette       1 cm light path                                                 Temperature   room temperature                                                Reading       against reagent blank for standard                                            and sample; against distilled water                                           for sample blank                                                ______________________________________                                    

    ______________________________________                                        Pipette into test tubes:                                                               Reagent  Sample                                                               blank    blank   Standard   Sample                                   ______________________________________                                        Distilled water                                                                          0.20       --      --       --                                     Sample     --         0.20 ml --       0.20 ml                                Standard   --         --      0.20 ml  --                                     Work, Solution                                                                           --         1.00 ml --       --                                     without indicator                                                             Work, Solution                                                                           1.00 ml    --      1.00 ml  1.00 ml                                ______________________________________                                    

Mix and allow to stand at room temperature for 5 min. Read theabsorbance of the sample blank (Asb) against distilled water and theabsorbance of the sample (As) and of the standard (Ast) against thereagent blank. ##EQU1##

OPTIMIZATION STUDIES pH Optimization

Starting with a formulation containing the following components:

    ______________________________________                                        Indicator     3.5    mmol/L                                                   Thiourea      63     mmol/L                                                   Ascorbic acid 10     mmol/L                                                   Buffer        180    mmol/L: pH range 0.5-5.0                                 ______________________________________                                    

The effect of pH on the iron test was studied using 3 aqueous solutionsof iron at concentrations of approximately 200, 500 and 1000 μg/dl andtwo different human plasma pools at approximately 300 μg/dl of iron.Different types of buffer were used to cover the pH range:

    KC1/HC1 for the pH 0.5-1.0-1.5-2.0

    Citric acid/NaOH for the pH 2.0-2.5-3.0

    Acetic acid/HaOH for the pH 3.0-4.5-5.0

Color development was monitored at 593 nm and the absorbance after 5min. at room temperature (end-point of the reaction) was taken (seeTable 1). The average absorbance for the three aqueous solutions of ironand for the two pools of plasma were calculated and plotted vs. pH (FIG.1)

From the data it is evident that the indicator compound according to theinvention can be used for pH values equal or greater than 1, andpreferably of about 1 to facilitate the release of iron fromtransferrin.

Choice of the buffer

Compounds able to give buffer solution at pH 1 were selected; e.g. usingthe KC1/HC1 or citric acid or malonic acid as buffer agents and testingat pH 1.0 and 0.3 mol/L aqueous solution of iron and human plasma. Nodifference in absorbance response and time of reaction were noticed. Allthe compounds tested were found to have the same buffer capacity withhuman sera.

Choice and Optimization of the Reducing Agent

Ascorbic acid is the reducing agent generally used to reduce ferricions; unfortunately the compound is stable only for a few hours when itis put into solution. Consequently, in general the iron kitscommercially available, supply the ascorbic acid in powder form to beadded manually to a preformed solution (i.e. see Sera-Pak Iron kit). Inorder to obtain a ready to use solution a search was carried out to finda more stable and appropriate reducing agent. The3-hydroxy-1,2,3,4-tetrahydro-benzo(h) quinoline (HTBQ) ##STR17## wasfound very suitable, in an acid medium, the ascorbic acid to reduce theferric ions to ferrous ions and to promote the iron release fromtransferrin.

Starting with a formulation containing the following components:

    ______________________________________                                        HCl/KCl buffer pH 1.0;                                                                             100    mmol/L                                            Indicator            3.5    mmol/L                                            Thiourea             63     mmol/L                                            ______________________________________                                    

The HTBQ was added in concentrations ranging from 0 to 25 mmol/L and theabsorbance response to 593 nm after 5 min of reaction was recorded usingaqueous solutions of iron and two different human plasma pools. Data inFIG. 2 show that a minimum amount of 5-10 mmol/L of HTBQ is required; aconcentration of about 10 mmol/L is preferable.

Indicator Optimization

Starting with a formulation containing:

    ______________________________________                                        HCl/KCl buffer pH 1.0;                                                                             200    mmol/L                                            Thiourea             63     mmol/L                                            HTBQ                 20     mmol/L                                            ______________________________________                                    

The indicator was added in concentrations ranging from 0.5 to 10 mmol/Land the absorbance response at 593 nm after 5 min. of reaction wasmonitored using aqueous solutions of iron and two different pools ofhuman plasma. Data in FIG. 3 show that a minimum amount of 2.5-3 mmol/Lof the indicator is required; a concentration of about 3.5 mmol/L ispreferable.

From the optimization studies carried out the following remarks can bemade:

1. pH: the system works in the pH range from 1 to 5; a pH=1 is preferredto facilitate the dissociation of iron from transferrin. Choosing a pHover 3.0 it is preferable to introduce in the working solution asurfactant to avoid possible sample turbidity; Triton X-100 or Tween 10at a concentration of 0.5% can be used. pH higher than 5 were not testedbut it is presumable that the system could work if an appropriatecomponent to dissociate iron from transferrin is used.

2. Buffer: different types of compounds can be used (e.g. citric acid,malonic acid, HC1/KC1).

3. Molarity; a buffer molarity less than 400 mmol/L and preferably ofabout 200 mmol/L is preferred to avoid possible human sample turbidity;however a molarity higher than 400 mmol/L can be used in appropriatesurfactants are used.

4. Reducing Agent: HTBQ can conveniently substitute the ascorbic acid. Aconcentration above 5 mmol/L is suggested, a concentration of about 20mmol/L is preferred.

5. Indicator Compound: a concentration above 2.5 mmol/L is suggested, aconcentration of about 3,5 mmol/L is preferred.

6. Thiourea: using a formulation at pH 1.0 this component is notnecessary and its use can be avoided; above this pH value aconcentration of 63 mmol/L is satisfactory to suppress copperinterference, (data not shown).

PERFORMANCE VALIDATION

The performance validation was carried out according to the testprocedure initially reported and with formulation containing thefollowing components:

    ______________________________________                                        HCl/KCl Buffer pH 1.0;                                                                             200    mmol/L                                            Indicator            3.5    mmol/L                                            HTBQ                 20     mmol/L                                            ______________________________________                                    

Linearity tests

Aqueous standards of ferric ions, prepared by dissolving iron metal (NBSmaterial) in nitric acid and diluted to appropriate concentrations withdistilled water, were assayed in triplicate. FIG. 4 shows a linearity upto at least 1000 μg/dl iron.

Comparison study

Comparative assays were conducted using the Sera-Pak Iron kit and thepresent formulation. 25 human plasma heparin were used as samples.

The results obtained, elaborated statistically by a linear method, areshown in FIG. 5. The correlation between the two methods is very good.

What is claimed is:
 1. Compounds of the general formula (I) ##STR18##wherein R¹ denotes hydrogen, halogen or C₁ -C₄ alkyl andR² denotes C₁-C₄ alkyl, aryl or heteroalkyl or a substituted C₁ -C₄ alkyl, aryl orheteroaryl.
 2. Compounds of formula (I) according to claim 1 whereinR¹denotes hydrogen and R² denotes C₁ -C₄ -alkyl, aryl or thienyl, whichradicals can be substituted by C₁ -C₄ -alkyl, hydrogen, halogen or -SO₃H.
 3. Compounds of formula (I) according to claim 1 whereinR¹ denoteshalogen and R² denotes methyl, ethyl, propyl, phenyl, tolyl or thienyl.4. Compound of formula (I) according to claim 1 whereinR¹ denoteshydrogen and R² denotes ##STR19## wherein R¹¹ denotes hydrogen, methylor chlorine.
 5. Use of a compound according to claim 1 in thedetermination of iron in a biological fluid by reflectance method.